The main therapeutic challenge in the treatment of myeloid leukemias is the development of strategies that maximize the induction of leukemia cell apoptosis before resistance to chemotherapy develops. p53 is the master switch that determines whether a stressed cell undergoes apoptosis, thus acting as a tumor suppressor. p53 mutations lead to inactivation of this suppressor function. Mouse Double Minute 2 (HDM2) and its homolog HDMX can also inactivate p53 activity: while HDM2 is an ubiquitin ligase that mediates degradation of p53 by ubiquitin-mediated proteolysis, HDMX inhibits the transcriptional activity of p53. p53 mutations leading to p53 inactivation are rare in newly diagnosed and relapsed AML. There is a reported loss of p53 function through over-expression of HDM2 in approximately 50% of AML cases; expression of HDMX have not been investigated in AML. Restoration of p53 activity by inhibiting HDM2/p53 interaction utilizing non-genotoxic small molecule inhibitors (Nutlin 3a, Ml 63) induces apoptosis in AML cells with unmutated p53. While these HDM2 inhibitors dramatically increase p53 levels that initiate transcription of p53 targets, transcription-independent direct interactions of p53 with Bcl-2 family members also occur. Furthermore, Chemotherapeutic agents such as cytarabine and daunorubicin syngergize with BH3 mimetics, and with MARK inhibitors, which inhibit induction of anti-apoptotic p21 and regulate the subcellular distribution of p53. Thus, we propose to investigate the molecular and clinical consequences of a clinical trial with small molecule inhibitors of HDM2 (Nutlin 3a, MI-63) and to develop a better understanding of the mechanisms regulating p53 activation and the observed synergism with chemotherapy. If successful, these studies will provide rationale for a novel therapeutic strategy in AML based on the non-genotoxic activation of p53 signaling. Specific Aim 1: Identify the molecular determinants of apoptosis induced by non-genotoxic small molecule inhibitors of HDM2 (Nutlin 3a, Ml 63) in leukemia cell lines, primary leukemia cells and stem cells. Specific Aim 2 Determine mechanisms by which HDM2 inhibition synergizes with chemotherapy (Ara- C, Doxorubicin) in AML. Specific Aim 3 Conduct first-in-man Phase I trial of HDM2 inhibitors (Nutlin 3a analog R7112, Ml 63) in AML Lay Language Summary: AML is a largely incurable malignancy of bone marrow stem cells. A gene termed p53, a master regulator of cell survival and death, is frequently dysregulated in cancers and leukemias. While cancers have a high frequency of p53 mutations, leukemias do not. This finding opens a window to activate the function of this gene by releasing p53 from its inhibitor, the HDM2 protein, by a novel class of drugs with great therapeutic promise. This proposal lays the groundwork for an entirely novel concept for the treatment of AML.